This invention pertains to the field of oncology. In particular, this invention pertains to the discovery of a tumor suppressor gene implicated in the etiology of nevoid basal cell carcinoma syndrome (NBCCS) and various cancers including basal cell carcinomas.
Many cancers are believed to result from a series of genetic alterations leading to progressive disordering of normal cellular growth mechanisms (Nowell (1976) Science 194:23, Foulds (1958) J. Chronic Dis. 8:2). In particular, the deletion or multiplication of copies of whole chromosomes or chromosomal segments, or specific regions of the genome are common (see, e.g., Smith et al. (1991) Breast Cancer Res. Treat. 18: Suppl. 1: 5-14; van de Vijer and Nusse (1991) Biochim. Biophys. Acta. 1072: 33-50; Sato et al. (1990) Cancer. Res. 50: 7184-7189). In particular, the amplification and deletion of DNA sequences containing proto-oncogenes and tumor-suppressor genes, respectively, are frequently characteristic of tumorigenesis. Dutrillaux et al. (1990) Cancer Genet. Cytogenet. 49: 203-217.
One cancer-related syndrome that appears to have a strong genetic base is the nevoid basal cell carcinoma syndrome (NBCCS). The nevoid basal cell carcinoma syndrome, also known as Gorlin syndrome and the basal cell nevus syndrome, is an autosomal dominant disorder that predisposes to both cancer and developmental defects (Gorlin (1995) Dermatologic Clinics 13: 113-125). Its prevalence has been estimated at 1 per 56,000, and 1-2% of medulloblastomas and 0.5% of basal cell carcinomas (BCCs) are attributable to the syndrome (Springate (1986) J. Pediatr. Surg. 21: 908-910; Evans et al. (1991) British J. Cancer. 64: 959-961). In addition to basal cell carcinomas (BCCs) and medulloblastomas, NBCCS patients are also at an increased risk for ovarian fibromas, meningiomas, fibrosarcomas, rhabdomyosarcomas, cardiac fibromas and ovarian dermoids (Evans et al. (1991) supra., Evans et al. (1993) J. Med. Genet. 30: 460-464; Gorlin (1995) supra.).
Non-neoplastic features, including odontogenic keratocysts (which are most aggressive in the second and third decades of life), pathognomonic dyskeratotic pittina of the hands and feet, and progressive intracranial calcification (usually evident from the second decade) are very common. There is a broad range of skeletal defects (Gorlin (1995) supra.; Shanley et al. (1994) Am. J. Med. Genet. 50: 282-290) including rib, vertebral and shoulder anomalies, pectus excavatum, immobile thumbs and polydactyly. Craniofacial and brain abnormalities include cleft palate, characteristic coarse fades, strabismus, dysgenesis of the corpus callosum macrocephaly and frontal bossing (Gorlin (1995) supra.). Generalized overgrowth (Bale et al. (1991) Am. J. Med. Genet. 40: 206-210) and acromegalic appearance are common, but growth hormone and IGF1 levels are not elevated.
Implications for the affected individual can be severe, predominantly due to the prolific basal cell carcinomas which can number more than 500 in a lifetime (Shanley et al. (1994) supra). Expression of many features of the syndrome is variable, but the severity tends to breed true within families (Anderson et al. (1967) Am. J. Hum. Genet., 19:12-22). This variation between families may reflect specific phenotypic effects of different mutations, modifier genes, or environmental factors (sunlight exposure is likely to play a role in the age of onset and incidence of basal cell carcinomas). One third to one half of patients have no affected relatives and are presumed to be the product of new germ cell mutations (Gorlin (1995) supra.). Unilateral and segmental NBCCS are attributed to somatic mutation in one cell of an early embryo (Gutierrez and Mora (1986) J. Am. Acad. Dermatol. 15: 1023-1029).
The NBCCS syndrome was mapped to one or more genes at chromosome 9q22-31 (Gailani et al. (1992) Cell 69: 111-117; Reis et al. (1992) Lancet 339: 617; Farndon et al. (1992) Lancet 339: 581-2). In addition, it has been demonstrated that the same region is deleted in a high percentage of basal cell carcinomas and other tumors related to the disorder (Gailani et al. (1992) supra.) thus suggesting that the NBCCS gene functions as a tumor suppressor. Inactivation of NBCCS gene(s) may be a necessary if not sufficient event for the development of basal cell carcinomas (Shanley et al. (1995) Hum. Mol. Genet. 4: 129-133; Gailani et al. (1996) J. Natl. Canc. Inst. 88: 349-354).
Since the original mapping of the gene in 1992, linkage studies have narrowed the NBCCS region to a 4 cM interval between D9S180 and D9S196 (Goldstein et al. (1994) Am. J. Hum. Genet. 54: 765-773; Wicking et al. (1994) Genomics 22: 505-511). Reported recombination involving an unaffected individual tentatively placed the gene proximal to D9S287 (Farndon et al. (1994) Genomics 23: 486-489). The 9q22 region, however, is very gene rich and appeared to contain at least two tumor suppressor genes. In addition, Harshman et al. (1995) Hum. Mol. Genet. 4: 1259-1266, showed that different methods of identifying cDNAs from a genomic region result in a surprisingly different array of candidate genes. Thus, prior to this invention the specific NBCCS gene was unknown.
This invention provides for a nucleic acid sequence (e.g., a cDNA) associated with nevoid basal cell carcinoma syndrome (NBCCS) and with various cancers including various sporadic basal cell carcinomas (BCCs). The NBCCS gene disclosed herein appears to be a tumor-suppressor gene and is a homologue of the Drosophila patched (ptc) gene. The human NBCCS gene is therefore also referred to herein as the human PATCHED (PTC) gene.
Absence, partial inactivation (e.g., through haploinsufficiency or mutation), complete inactivation, or otherwise altered expression of the NBCCS (PTC) gene causes or creates a predisposition to NBCCS and/or to the onset of basal cell carcinomas.
In one preferred embodiment, this invention provides an isolated human nucleic acid encoding a nevoid basal cell carcinoma syndrome (NBCCS) (PTC) protein, wherein said nucleic acid specifically hybridizes, under stringent conditions, to a second nucleic acid consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NOS. 1, 58 and 59, in the presence of a human genomic library under stringent conditions. The isolated nucleic acid is at least 30, preferably at least 50, more preferably at least 100, and most preferably at least 200 nucleotides in length.
In another embodiment, the isolated NBCCS nucleic acid has at least 75 percent sequence identity, preferably at least 85 percent, sequence identity, more preferably at least 90% sequence identity and most preferably at least 95 percent or even at least 98% sequence identity across a window of at least 30 nucleotides, preferably across a window of at least 50 nucleotides, more preferably across a window of at least 80 nucleotides, and most preferably across a window of at least 100 nucleotides, 200 nucleotides, 500 nucleotides or even the full length with the nucleic acid of SEQ ID NOS: 1, 58, or 59.
In one embodiment, the isolated human NBCCS nucleic acid is amplified from a genomic library using any of the primer pairs provided in Table 2. In another embodiment, the NBCCS nucleic acid is identified by specific hybridization with any of the nucleic acids amplified from a genomic library using any of the primer pairs provided in Table 2. In a particularly preferred embodiment the nucleic acid is a nucleic acid selected from the group consisting of SEQ ID NO: 1, SEQ ID NOS: 1, 58 and 59.
In another embodiment, this invention provides for an isolated human nevoid basal cell carcinoma syndrome (NBCCS) (PTC) nucleic acid sequence, wherein said nucleic acid encodes a polypeptide subsequence of at least 10 contiguous amino acid residues of the polypeptide encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 58 and 59, or conservative substitutions of said polypeptide subsequence. The isolated human NBCCS nucleic acid is preferably at least 50, more preferably at least 100, and most preferably at least 200, 400, 500, or even 800 residues (amino acids) in length. In a particularly preferred embodiment, the nucleic acid encodes a polypeptide sequence encoded by a nucleic acid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 58, and SEQ. ID NO: 59. Even more preferably, the NBCCS nucleic acid is a nucleic acid selected from the group consisting of SEQ ID NOS: 1, 58 and 59.
In still yet another embodiment, this invention provides an isolated nucleic acid encoding a human nevoid basal cell carcinoma (NBCCS) (PTC) polypeptide comprising at least 10 contiguous amino acids from a polypeptide sequence encoded by a nucleic acid selected from the group consisting SEQ ID NOS: 1, 58 and 59, wherein: said polypeptide, when presented as an antigen, elicits the production of an antibody which specifically binds to a polypeptide sequence encoded by a nucleic acid selected from the group consisting of SEQ ID NOS: 1, 58 and 59, and said polypeptide does not bind to antisera raised against a polypetide encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 58 and 59 which has been fully immunosorbed with a polypeptide encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 58 and 59. Even more preferably this NBCCS nucleic acid hybridizes to a clone of the human PTC gene present in a human genomic library under stringent conditions and even more preferably hybridizes to a nucleic acid selected from the group consisting of SEQ ID NOS: 1, 58 and 59.
The invention also provides isolated nucleic acids that include one or more mutations compared to a nucleic acid selected from the group consisting of SEQ ID NOS: 1, 58 and 59. The mutations can be, for example, missense mutations, nonsense mutations, frameshift mutations, and splicing mutations. Alternatively, the mutations can be in regulatory regions that affect expression of the NBCCS gene.
In another embodiment, this invention provides for vectors incorporating any of the above-described nucleic acids. The vectors preferably include the above-described nucleic acid operably linked (under the control of) a promoter; either constitutive or inducible. The vector can also include an initiation and a termination codon.
This invention also provides for an isolated human NBCCS (PTC) polypeptide, said polypeptide comprising a subsequence of at least 10 contiguous amino acids of a polypeptide encoded by a nucleic acid selected from the group consisting of SEQ ID NOS: 1, 58 and 59, or conservative substitutions of said polypeptide subsequence. This NBCCS polypeptide is preferably at least 50, more preferably at least 100, and most preferably at least 200, 400, 500, or even 800 residues (amino acids) in length. The polypeptide can be a polypeptide encoded by a nucleic acid amplified from genomic DNA or an RNA using any of the primers pairs provided in Table 2. In a particularly preferred embodiment, the NBCCS polypeptide is a polypeptide encoded by a nucleic acid selected from the group consisting of SEQ ID NOS: 1, 58 and 59.
This invention also includes an isolated NBCCS (PTC) polypeptide comprising at least 10 contiguous amino acids from a polypeptide sequence encoded by a nucleic acid selected from the group consisting of SEQ ID NOS: 1, 58 and 59, wherein: said polypeptide, when presented as an antigen, elicits the production of an antibody which specifically binds to a polypeptide encoded by a nucleic acid selected from the group consisting of SEQ ID NOS: 1, 58 and 59, and said polypeptide does not bind to antisera raised against a polypeptide encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 58 and 59 which has been fully immunosorbed with a polypeptide encoded by a sequence selected from the group consisting of SEQ ID NOS: 1, 58 and 59. This polypeptide is preferably at least 50, more preferably at least 100, and most preferably at least 200, 400, 500, or even 800 amino acid residues in length. In a particularly preferred embodiment, this polypeptide is encoded by a nucleic acid selected from the group consisting of SEQ ID NOS: 1, 58 and 59.
The polypeptides of this invention can include conservative substitutions of any of the above-described polypeptides. In a particularly preferred embodiment the above-described nucleic acids and/or proteins, or subsequences thereof, are not a PTC nucleic acid or polypeptide from a Drosophila, a murine, or C. elegans. 
In another embodiment, this invention provides for anti-NBCCS antibodies. Particularly preferred antibodies specifically bind a polypeptide comprising at least 10, more preferably at least 20, 40, 50, and most preferably at least 100, 200, 400, and even 800 contiguous amino acids, or even the full length polypeptide encoded by a nucleic acid selected from the group consisting of SEQ ID NOS: 1, 58 and 59 wherein: said polypeptide, when presented as an antigen, elicits the production of an antibody which specifically binds to a polypeptide encoded by a nucleic acid selected from the group consisting of SEQ ID NOS: 1, 58 and 59, and said polypeptide does not bind to antisera raised against a polypeptide encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 58 and 59 which has been fully immunosorbed with a polypeptide encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 58 and 59. The antibody can be polyclonal or monoclonal. The antibody can also be humanized or human.
This invention also provides for cells (e.g., recombinant cells such as hybridomas or triomas) expressing any of the above-described antibodies.
This invention also provides for methods of detecting a predisposition to nevoid basal cell carcinoma syndrome (NBCCS) or to a basal cell carcinoma. The methods include the steps of i) providing a biological sample of the organism; and ii) detecting a human NBCCS (PTC) gene or gene product in the sample. The provision of a biological sample and detection methods are described herein. In particular, detecting can involve detecting the presence or absence, or quantifying an NBCCS gene or subsequence thereof including any of the above-described nucleic acids. The detecting can also involve detecting the presence or absence or quantifying a NBCCS polypeptide or subsequence thereof including any of the above-described polypeptides. The detecting can involve detecting the presence or absence of normal or abnormal NBCCS nucleic acids or polypeptides. For example, one can detect a predisposition to BCC or NBCCS by detecting the presence of a mutation in a NBCCS nucleic acid. Particularly preferred assays include hybridization assays and/or sequencing for nucleic acids and immunoassays for NBCCS polypeptides.
In another embodiment, this invention provides for pharmacological compositions comprising a pharmaceutically acceptable carrier and a molecule selected from the group consisting of an vector encoding an NBCCS polypeptide or subsequence thereof, an NBCCS polypeptide or subsequence thereof, and an anti-NBCCS antibody as described herein.
This invention also provides for primers for the amplification of one or more exons of the NBCCS (PTC) gene. These primers include, but are not limited to the primers provided in Table 2.
This invention also provides kits for the detection and/or quantification of NBCCS gene or gene product. The kits can include a container containing one or more of any of the above identified nucleic acids, amplification primers, and antibodies with or without labels, free, or bound to a solid support as described herein. The kits can also include instructions for the use of one or more of these reagents in any of the assays described herein.
Finally, this invention also provides therapeutic methods. These include a methods of treating basal cell carcinoma and/or nevoid basal cell carcinoma syndrome and/or solar keratoses in a mammal. The methods can involve transfecting cells of the mammal with a vector expressing a nevoid basal cell carcinoma syndrome (NBCCS) polypeptide such that the cells express a functional NBCCS polypeptide as described herein. The transfection can be in vivo or ex vivo. Ex vivo transfection is preferably followed by re-infusion of the cells back into the organism as described herein. Other methods involve administering to the mammal a therapeutically effective dose of a composition comprising a NBCCS (PTC) polypeptide and a pharmacological excipient as described herein. The methods are preferably performed on mammals such as mice, rats, rabbits, sheep, goats, pigs, more preferably on primates including human patients.
The term xe2x80x9cantibodyxe2x80x9d refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one xe2x80x9clightxe2x80x9d (about 25 kD) and one xe2x80x9cheavyxe2x80x9d chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
Antibodies exist e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)xe2x80x22 a dimer of Fab which itself is a light chain joined to VHxe2x88x92CH1 by a disulfide bond. The F(ab)xe2x80x22 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)xe2x80x22 dimer into an Fabxe2x80x2 monomer. The Fabxe2x80x2 monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunology, Third Edition, W. E. Paul, ed., Raven Press, N.Y. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv).
An xe2x80x9canti-NBCCsxe2x80x9d antibody is an antibody or antibody fragment that specifically binds a polypeptide encoded by the NBCCS gene, cDNA, or subsequence thereof.
A xe2x80x9cchimeric antibodyxe2x80x9d is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
The term xe2x80x9cimmunoassayxe2x80x9d is an assay that utilizes an antibody to specifically bind an analyte. The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the analyte.
The terms xe2x80x9cisolatedxe2x80x9d xe2x80x9cpurifiedxe2x80x9d or xe2x80x9cbiologically purexe2x80x9d refer to material which is substantially or essentially free from components which normally accompany it as found in its native state.
The term xe2x80x9cnucleic acidxe2x80x9d refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides.
The terms xe2x80x9cpolypeptidexe2x80x9d, xe2x80x9cpeptidexe2x80x9d and xe2x80x9cproteinxe2x80x9d are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
A xe2x80x9clabelxe2x80x9d is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, dioxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available (e.g., the peptide of SEQ ID NO:1 can be made detectable, e.g., by incorporating a radio-label into the peptide, and used to detect antibodies specifically reactive with the peptide).
As used herein a xe2x80x9cnucleic acid probexe2x80x9d is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. As used herein, a probe may include natural (i.e. A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization. Thus, for example, probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages. It will be understood by one of skill in the art that probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. The probes are preferably directly labeled as with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled such as with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence.
A xe2x80x9clabeled nucleic acid probexe2x80x9d is a nucleic acid probe that is bound, either covalently, through a linker, or through ionic, van der Waals or hydrogen bonds to a label such that the presence of the probe may be detected by detecting the presence of the label bound to the probe.
The term xe2x80x9ctarget nucleic acidxe2x80x9d refers to a nucleic acid (often derived from a biological sample), to which a nucleic acid probe is designed to specifically hybridize. It is either the presence or absence of the target nucleic acid that is to be detected, or the amount of the target nucleic acid that is to be quantified. The target nucleic acid has a sequence that is complementary to the nucleic acid sequence of the corresponding probe directed to the target. The term target nucleic acid may refer to the specific subsequence of a larger nucleic acid to which the probe is directed or to the overall sequence (e.g., gene or mRNA) whose expression level it is desired to detect. The difference in usage will be apparent from context.
xe2x80x9cSubsequencexe2x80x9d refers to a sequence of nucleic acids or amino acids that comprise a part of a longer sequence of nucleic acids or amino acids (e.g., polypeptide) respectively.
The term xe2x80x9crecombinantxe2x80x9d when used with reference to a cell, or nucleic acid, or vector, indicates that the cell, or nucleic acid, or vector, has been modified by the introduction of a heterologous nucleic acid or the alteration of a native nucleic acid, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
The term xe2x80x9cidenticalxe2x80x9d in the context of two nucleic acids or polypeptide sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection.
An additional algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which is described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra.). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=xe2x88x924, and a comparison of both strands.
The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see, e.g., Karlin and Altschul (1993) Proc. Nat""l. Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to an NBCCS gene or cDNA if the smallest sum probability in a comparison of the test nucleic acid to an NBCCS nucleic acid (e.g., SEQ ID NOS: 1, 58 or 59) is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
The term xe2x80x9csubstantial identityxe2x80x9d or xe2x80x9csubstantial similarityxe2x80x9d in the context of a polypeptide indicates that a polypeptide comprises a sequence with at least 70% sequence identity to a reference sequence, or preferably 80%, or more preferably 85% sequence identity to the reference sequence, or most preferably 90% identity over a comparison window of about 10-20 amino acid residues. An indication that two polypeptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution.
An indication that two nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.
xe2x80x9cBind(s) substantiallyxe2x80x9d refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target polynucleotide sequence.
The phrase xe2x80x9chybridizing specifically toxe2x80x9d, refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. The term xe2x80x9cstringent conditionsxe2x80x9d refers to conditions under which a probe will hybridize to its target subsequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. (As the target sequences are generally present in excess, at Tm, 50% of the probes are occupied at equilibrium). Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30xc2x0 C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60xc2x0 C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
The phrases xe2x80x9cspecifically binds to a proteinxe2x80x9d or xe2x80x9cspecifically immunoreactive withxe2x80x9d, when referring to an antibody refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind preferentially to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to a protein under such conditions requires an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
A xe2x80x9cconservative substitutionxe2x80x9d, when describing a protein refers to a change in the amino acid composition of the protein that does not substantially alter the protein""s activity. Thus, xe2x80x9cconservatively modified variationsxe2x80x9d of a particular amino acid sequence refers to amino acid substitutions of those amino acids that are not critical for protein activity or substitution of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitutions of even critical amino acids do not substantially alter activity. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
See also, Creighton (1984) Proteins, W. H. Freeman and Company. In addition, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are also xe2x80x9cconservatively modified variationsxe2x80x9d.
The terms human xe2x80x9cPTCxe2x80x9d or human xe2x80x9cNBCCS gene or cDNAxe2x80x9d are used interchangeably to refer to the human homologue of the Drosophila patched (ptc) gene disclosed herein. As explained below, the human PTC gene is a tumor suppressor gene also involved in the etiology of nevoid basal carcinoma cell syndrome.
A xe2x80x9cgene productxe2x80x9d, as used herein, refers to a nucleic acid whose presence, absence, quantity, or nucleic acid sequence is indicative of a presence, absence, quantity, or nucleic acid composition of the gene. Gene products thus include, but are not limited to, an mRNA transcript, a cDNA reverse transcribed from an mRNA, an RNA transcribed from that cDNA, a DNA amplified from the cDNA, an RNA transcribed from the amplified DNA or subsequences of any of these nucleic acids. Polypeptides expressed by the gene or subsequences thereof are also gene products. The particular type of gene product will be evident from the context of the usage of the term.
An xe2x80x9cabnormal PTC (or NBCCS) gene or cDNAxe2x80x9d refers to a NBCCS gene or cDNA that encodes a non-functional NBCCS polypeptide, or an NBCCS polypeptide of substantially reduced functionality. Non-functional, or reduced functionality, NBCCS polypeptides are characterized by a predisposition (i.e., an increased likelihood as compared to the xe2x80x9cnormalxe2x80x9d population) for, or the onset of, nevoid basal cell carcinoma syndrome. Similarly, xe2x80x9cabnormal PTC (or NBCCS) gene productxe2x80x9d refers to a nucleic acid encoding a non-functional or reduced functionality NBCCS polypeptide or the non-functional or reduced functionality NBCCS polypeptide itself. Abnormal NBCCS (PTC) genes or gene products include, for example, NBCCS genes or subsequences altered by mutations (e.g. insertions, deletions, point mutations, etc.), splicing errors, premature termination codons, missing initiators, etc. Abnormal NBCCS polypeptides include polypeptides expressed by abnormal NBCCS genes or nucleic acid gene products or subsequences thereof. Abnormal expression of NBCCS genes includes underexpression (as compared to the xe2x80x9cnormalxe2x80x9d healthy population) of NBCCS e.g., through partial or complete inactivation, haploinsufficiency, etc.